[go: up one dir, main page]

US6057308A - Remedy or preventive for hyperlipemia - Google Patents

Remedy or preventive for hyperlipemia Download PDF

Info

Publication number
US6057308A
US6057308A US09/180,745 US18074598A US6057308A US 6057308 A US6057308 A US 6057308A US 18074598 A US18074598 A US 18074598A US 6057308 A US6057308 A US 6057308A
Authority
US
United States
Prior art keywords
group
taurine
oryzanol
hyperlipemia
lipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
US09/180,745
Inventor
Hideaki Kitajima
Kenji Tsunoda
Teruyoshi Yanagita
Shigeru Murakami
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Taisho Pharmaceutical Co Ltd
Original Assignee
Taisho Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Taisho Pharmaceutical Co Ltd filed Critical Taisho Pharmaceutical Co Ltd
Assigned to TAISHO PHARMACEUTICAL CO., LTD. reassignment TAISHO PHARMACEUTICAL CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KITAJIMA, HIDEAKI, MURAKAMI, SHIGERU, TSUNODA, KENJI, YANAGITA, TERUYOSHI
Application granted granted Critical
Publication of US6057308A publication Critical patent/US6057308A/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to a pharmaceutical reparation for treating or preventing hyperlipemia.
  • Blood lipid components such as cholesterols, neutral fats, phospholipids or free fatty acids are in the form of water-soluble lipoproteins in the blood, which are classified into very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) by the difference of density.
  • VLDL very-low-density lipoprotein
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • Hyperlipemia is in a status of higher blood concentration of these lipid components which is caused by abnormal lipoprotein metabolism in the body than the normal concentration.
  • Taurine (aminoethylsulfonic acid) is a sulfur-containing amino acid having a very simple chemical structure with a molecular weight of 125.14, and known to have various pharmacological properties, over a broad area beginning with central nervous system, circulatory system and hepatobiliary system. Concerning the properties of taurine to the cholesterol metabolism, there are also known the lowering effect of serum cholesterol via cholagogic function and inhibition of gallstone formation by cholesterol.
  • ⁇ -Oryzanol is known to have the properties to inhibit the absorption of cholesterol and improve the lipoprotein metabolism.
  • dietetic therapy is first applied, and when it is not sufficiently effective, drug theraphy needs to be applied.
  • a desired drug for preventing the origination of hyperlipemia is one which maintains the low blood concentration of lipid components (e.g. cholesterols, neutral fats, phospholipids and free fatty acids), has no side-effect, and can be administered safely for a long term.
  • lipid components e.g. cholesterols, neutral fats, phospholipids and free fatty acids
  • An object of the present invention is to provide a pharmaceutical preparation of treating or preventing hyperlipemia which is safe even if applied for a long term.
  • the present inventors have found that an administration of taurine and ⁇ -oryzanol at the same time has a greater effect to improve the metabolism of excess amount of lipid components in the body such as cholesterol than an administration of either taurine or ⁇ -oryzanol alone, and have accomplished the present invention by the finding.
  • the present invention relates to a pharmaceutical preparation for treating or preventing hyperlipemia comprising taurine and ⁇ -oryzanol as effective components.
  • the pharmaceutical preparation for treating or preventing hyperlipemia of the present invention lowers the blood concentration of excess amount of lipid components in the body (e.g. cholesterols, neutral fats, phospholipids and free fatty acids), and has a property to improve lipoprotein metabolism, thereby it is useful as a pharmaceutical preparation for treating or preventing hyperlipemia, especially, it is very useful as a pharmaceutial preparation for preventing hyperlipemia because it has no side-effect and can be administered safely for a long term.
  • lipid components in the body e.g. cholesterols, neutral fats, phospholipids and free fatty acids
  • the effective dose of taurine is from 100 mg to 6000 mg for a healthy adult per day, preferably 1000 mg to 3000 mg.
  • the effective dose of ⁇ -oryzanol is from 1 mg to 6000 mg for a healthy adult per day, preferably 5 mg to 500 mg.
  • the amount ratio of taurine to ⁇ -oryzanol is 300 : 1 ⁇ 30.
  • the pharmaceutical preparation for treating or preventing hyperlipemia of the present invention can 15 contain other known additives, if desired, for example, fillers, disintegrators, binders, lubricants, anti-oxidants, coating agents, coloring agents, corrigents, surfactants, plasticizers and like, and can be formulated in the form of granules, powders, capsules, tablets, dry-syrups or solutions according to an ordinary method.
  • the pharmaceutical preparation of the present invention containing taurine and ⁇ -oryzanol as effective components is more effective to treat or prevent hyperlipemia by further addition of pantethine and nicotinic acid thereto.
  • one or more vitamins, other physiologically effective components, hormones, nutrient components, perfumes and the like can be added.
  • the pharmaceutical preparation of the present invention containing taurine and ⁇ -oryzanol as effective components lowers the blood concentration of cholesterols and neutral fats, and has a property to improve lipoprotein metabolism, therefore, it is useful as a pharmaceutical preparation for treating or preventing hyperlipemia, and especially, it is very useful as a pharmaceutical preparation for preventing hyperlipemia because it has no side-effect and can be administered safely for a long term.
  • Example 1 treated group Each group of rats was treated with the granules obtained in Example 1 per day (Example 1 treated group), 3000 mg of taurine (Taurine treated group), 300 mg of ⁇ -Oryzanol ( ⁇ -Oryzanol treated group) and not treated with any drug (Fat-enriched diet group).
  • the fat-enriched diet refers to a diet which is obtained by adding 5% cholesterol, 2% cholic acid and 20% beef tallow to a basal diet.
  • the basal diet is obtained from the following composition wherein the vitamin mixture and mineral mixture were the same as described by Harper (J. Nutr., 67, 109 (1959)).
  • Each group of rats was fed the diet and was bred for 45 days. After the feeding, the blood was collected via the caudal vein of animals to measure the serum cholesterol concentration. After the end of breeding, the liver was excised to measure the cholesterol concentration.
  • the mesenteric artery was excised, the surrounding fat was stripped and dyed with Sudan III, and number of fat depositions was counted by microscopy to determine the inhibition rate of fat deposition of the mesenteric artery.
  • the serum cholesterol concentrations are shown in Table 1.
  • the serum cholesterol concentration in Example 1 treated group was kept lower than that in the other groups.
  • the inhibition rate of the liver cholesterol is calculated from the liver cholesterol concentration according to the following formula: ##EQU1##
  • the liver cholesterol concentration and the inhibition rate of liver cholesterol are shown in Table 2.
  • the liver cholesterol concentration in Example 1 treated group was kept low and the inhibition rate of the fat deposition in Example 1 treated group was high, in comparison with the other groups.
  • the deposition of fat was judged by four stages of the number of depositions as 0 to 20, 20 to 100, 100 to 200 and more than 200, which were scored to 1, 2, 3 and 4, respectively, an average score of each group was calculated, and the inhibition rate of the fat deposition of the mesenteric artery in each group is calcurated according to the following formula. ##EQU2##
  • Table 3 shows average scores of the fat deposition in each group and the inhibition rate of the fat deposition in the mesenteric artery.
  • the inhibition rate of the fat deposition in Example 1 treated group was high in comparison with the other groups.
  • T+ ⁇ Group is treated with 2.0 mM taurine and 0.05 mM ⁇ -Oryzanol, T group is treated with 2.0 mM taurine only, and C group is untreated with any drug as a control group.
  • DMEM Dulbecco's Modified Eagle's medium
  • LPDS lipoprotein deficient serum
  • DMEM Dulbecco's Modified Eagle's medium
  • Taurine was dissolved in phosphate-buffered saline (PBS), sterilized by filtration and added to the basal medium to be made up to the concentration of 2.0 mM to give a test medium.
  • PBS phosphate-buffered saline
  • ⁇ -Oryzanol was dissolved in dimethyl sulfoxide (DMSO) to be made up to the concentration of 0.05 mM, and added to the basal medium.
  • DMSO dimethyl sulfoxide
  • Hep G2 cells (Wister Institute) were precultured in DMEM (low glucose, 1500 mg/L) containing 10% FCS, penicillin (1 ⁇ 10 5 units/L) and streptomycin (100 mg/L), and were cultured at 37° C. in an atmosphere containing 95% air and 5% carbon dioxide. The medium was renewed at an interval of 2 to 3 days. After being confluent, the Hep G2 cells were subcultured as follows: after removing the medium in the dish, the Hep G2 cells were washed quickly with 0.25% trypsin solution (containing 0.1% EDTA), and after addition of a suitable amount of a trypsin solution, incubated at 37° C. for about 5 minutes.
  • DMEM low glucose, 1500 mg/L
  • FCS penicillin (1 ⁇ 10 5 units/L
  • streptomycin 100 mg/L
  • the Hep G2 cells were planted well and centrifuged (1000 rpm, 5 minutes) for recovery.
  • the cells were divided into 1/2-1/3 and were cultured by using a DMEM medium containing 10% FCS.
  • the number of the cells was counted using a Sitemeter.
  • the Hep G2 cells were planted in dishes (3.5 cm) (85 ⁇ 10 4 cells/dish) and were cultured by using a DMEM medium containing 1 ml of high glucose (4500 mg/ml) per dish.
  • the Hep G2 cells were made confluent (about 100 ⁇ 10 4 /dish), after which the medium was renewed by the experimental medium, followed by culturing for 24 hours.
  • the Hep G2 cells were washed once with PBS, and after addition of 1 ml of PBS, recovered using a policeman.
  • 0.5 ⁇ Ci of [ 3 H]glycerol or 0.5 ⁇ Ci of [ 14 C]acetic acid per dish as a radioactive lipid precursor was added before beginning the experiment.
  • Lipid in the cells and the medium were extracted according to the method of Bligh and Dyer.
  • the recovered Hep G2 cells were melted and crushed by using a sonicator (Sonifier 250, Bronson Corp.) to give cell homogenate.
  • a part of the cell homogenate was taken into a spitz tube with a screw cap, 3 ml of methanol and 1.5 ml of chloroform were added thereto, followed by stirring well.
  • the mixture was heated at 37° C. for 40 minutes, and 1.5 ml of chloroform and 1.6 ml of water were added thereto to give a mixture wherein the ratio of chloroform : methanol : water was 1:1:0.8, which was then centrifuged (3000 rpm, 15 minutes).
  • the lower-layer chloroform was recovered, concentrated under a nitrogen gas atmosphere and dissolved in a definite amount of petroleum ether, and lipid in the cells was extracted. Lipid in the medium was also extracted in the same manner.
  • the extracted lipid was fractionated into free cholesterol, cholesterol ester, diglyceride, triglyceride, etc. by using a silica gel G (0.25 mm in thickness)-thin layer chromatography (TLC) developing with petroleum ether : diethyl ether : acetic acid (82:18:1). After completion of the development, each of lipid fractions was detected by coloring with iodide vapor. After the fractionation with TLC, the silica gel of each fraction on the plate was taken into a vial for measurement, dissolved in Sintisol EX-H (Dojin Chemical Co.), and the radioactivity in each fraction was measured with a liquid scintillation counter (Wallac 1410, Pharmacia Co.). Correction was made for the background radioactivity and for quenching by the silica gel.
  • TLC silica gel G (0.25 mm in thickness)-thin layer chromatography
  • a part of the cell homogenate was used for determination of the protein according to the method of Lowry.
  • Table 4 shows effect on the excretion of the labeled lipid (cholesterol ester, free cholesterol and triglyceride) after 24 hours by incorporating [ 14 C]acetic acid into Hep G2 cell lipid medium.
  • the excretion of the labeled lipid in T+ ⁇ group was more significantly inhibited than that in the other groups.
  • Table 5 shows effect on the excretion of the labeled lipid (total lipid, triglyceride and diglyceride) after 24 hours by incorporating [ 3 H]glycerol into Hep G2 cell lipid medium.
  • the excretion of the labeled lipid in T+ ⁇ group was more significantly inhibited than that in the other groups.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

PCT No. PCT/JP96/01443 Sec. 371 Date Nov. 17, 1998 Sec. 102(e) Date Nov. 17, 1998 PCT Filed May 29, 1996 PCT Pub. No. WO97/45112 PCT Pub. Date Dec. 4, 1997[Object] : To provide a pharmaceutical preparation for treating or preventing hyperlipemia which lowers cholesterols and neutral fats in blood and has a property to improve lipoprotein metabolism. [Construction] : A pharmaceutical preparation for treating or preventing hyperlipemia comprising taurine and gamma -oryzanol as effective components.

Description

This application is a 371 of PCT/JP96/01443, filed May 29, 1996.
TECHNICAL FIELD
The present invention relates to a pharmaceutical reparation for treating or preventing hyperlipemia.
BACKGROUND ART
Blood lipid components such as cholesterols, neutral fats, phospholipids or free fatty acids are in the form of water-soluble lipoproteins in the blood, which are classified into very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) by the difference of density. Hyperlipemia is in a status of higher blood concentration of these lipid components which is caused by abnormal lipoprotein metabolism in the body than the normal concentration. When the status of hyperlipemia is continued for a long term, cholesterol and the like tend to deposit into the artery wall, and it has been found by many investigations and epidemiological research that atherosclerosis such as myocardial infarction, angina pectoris or cerebral infarction originates and develops. Accordingly, it is important to lower the blood concentration of these lipid components to the normal concentration for treating hyperlipemia, and it is important to maintein the normal concentration for preventing hyperlipemia.
Taurine (aminoethylsulfonic acid) is a sulfur-containing amino acid having a very simple chemical structure with a molecular weight of 125.14, and known to have various pharmacological properties, over a broad area beginning with central nervous system, circulatory system and hepatobiliary system. Concerning the properties of taurine to the cholesterol metabolism, there are also known the lowering effect of serum cholesterol via cholagogic function and inhibition of gallstone formation by cholesterol.
γ-Oryzanol is known to have the properties to inhibit the absorption of cholesterol and improve the lipoprotein metabolism.
For treating hyperlipemia at the present time, dietetic therapy is first applied, and when it is not sufficiently effective, drug theraphy needs to be applied.
Recently, there are some pharmaceutical drugs of hyperlipemia with different mechanism of action (absorption inhibition, excretion acceleration and synthesis inhibition), but they have a problem of the side-effects, main examples of which are leukocyte decrease, hepatic enlargement, gastrointestinal troubles, sexuality reduction, etc. Accordingly, a pharmaceutical drug for treating or preventing hyperlipemia without side-effects is desirable.
In view of preventive medicine, a desired drug for preventing the origination of hyperlipemia is one which maintains the low blood concentration of lipid components (e.g. cholesterols, neutral fats, phospholipids and free fatty acids), has no side-effect, and can be administered safely for a long term.
An object of the present invention is to provide a pharmaceutical preparation of treating or preventing hyperlipemia which is safe even if applied for a long term.
DISCLOSURE OF THE INVENTION
The present inventors have found that an administration of taurine and γ-oryzanol at the same time has a greater effect to improve the metabolism of excess amount of lipid components in the body such as cholesterol than an administration of either taurine or γ-oryzanol alone, and have accomplished the present invention by the finding.
Accordingly, the present invention relates to a pharmaceutical preparation for treating or preventing hyperlipemia comprising taurine and γ-oryzanol as effective components.
The pharmaceutical preparation for treating or preventing hyperlipemia of the present invention lowers the blood concentration of excess amount of lipid components in the body (e.g. cholesterols, neutral fats, phospholipids and free fatty acids), and has a property to improve lipoprotein metabolism, thereby it is useful as a pharmaceutical preparation for treating or preventing hyperlipemia, especially, it is very useful as a pharmaceutial preparation for preventing hyperlipemia because it has no side-effect and can be administered safely for a long term.
According to the present invention, the effective dose of taurine is from 100 mg to 6000 mg for a healthy adult per day, preferably 1000 mg to 3000 mg. The effective dose of γ-oryzanol is from 1 mg to 6000 mg for a healthy adult per day, preferably 5 mg to 500 mg. The amount ratio of taurine to γ-oryzanol is 300 : 1˜30.
The pharmaceutical preparation for treating or preventing hyperlipemia of the present invention can 15 contain other known additives, if desired, for example, fillers, disintegrators, binders, lubricants, anti-oxidants, coating agents, coloring agents, corrigents, surfactants, plasticizers and like, and can be formulated in the form of granules, powders, capsules, tablets, dry-syrups or solutions according to an ordinary method.
The pharmaceutical preparation of the present invention containing taurine and γ-oryzanol as effective components is more effective to treat or prevent hyperlipemia by further addition of pantethine and nicotinic acid thereto.
Furthermore, if desired, one or more vitamins, other physiologically effective components, hormones, nutrient components, perfumes and the like can be added.
INDUSTRIAL APPLICABILITY
The pharmaceutical preparation of the present invention containing taurine and γ-oryzanol as effective components lowers the blood concentration of cholesterols and neutral fats, and has a property to improve lipoprotein metabolism, therefore, it is useful as a pharmaceutical preparation for treating or preventing hyperlipemia, and especially, it is very useful as a pharmaceutical preparation for preventing hyperlipemia because it has no side-effect and can be administered safely for a long term.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is illustrated in more detail by the following examples and experiments.
EXAMPLE 1 
______________________________________                                    
Taurine                3000 mg                                            
γ-Oryzanol       10 mg                                              
Powder sugar           600 mg                                             
Aerosil                36 mg                                              
Aspartame              9 mg                                               
Low substituted        54 mg                                              
hydroxypropyl cellulose                                                   
______________________________________
Following an ordinary method, there were obtained granules containing the above-mentioned components.
EXAMPLE 2 
______________________________________                                    
Taurine                3000 mg                                            
γ-Oryzanol       10 mg                                              
Vitamin E              100 mg                                             
Powder sugar           600 mg                                             
Aerosil                600 mg                                             
Aspartame              9 mg                                               
Low substituted        54 mg                                              
hydroxypropyl cellulose                                                   
Magnesium stearate     24 mg                                              
Pigment                trace                                              
Perfume                trace                                              
______________________________________
Following an ordinary method, there were obtained granules containing the above-mentioned components.
EXAMPLE 3 
______________________________________                                    
Taurine                3000 mg                                            
Pantethine             50 mg                                              
γ-Oryzanol       10 mg                                              
Powder sugar           600 mg                                             
Aerosil                36 mg                                              
Aspartame              9 mg                                               
Low substituted        54 mg                                              
hydroxypropyl cellulose                                                   
______________________________________
Following an ordinary method, there were obtained granules containing the above-mentioned components.
EXAMPLE 4 
______________________________________                                    
Taurine                3000 mg                                            
γ-Oryzanol       10 mg                                              
Vitamin E              100 mg                                             
Vitamin B.sub.1        10 mg                                              
Vitamin B.sub.2        20 mg                                              
Vitamin B.sub.6        10 mg                                              
Vitamin C              600 mg                                             
Nicotinic acid         10 mg                                              
Pantethine             50 mg                                              
Starch                 600 mg                                             
Low substituted        12 mg                                              
hydroxypropyl cellulose                                                   
Aspartame              12 mg                                              
Magnesium stearate     24 mg                                              
______________________________________
Following an ordinary method, there were obtained granules containing the above-mentioned components.
Experiment 1: Effect on SHRSP (spontaneously hypertensive rats stroke-prone) by fat-enriched diet
[Experimental Animals]
Six male SHRSP, 3 months of age, were used for each group.
[Experimental Group]
Each group of rats was treated with the granules obtained in Example 1 per day (Example 1 treated group), 3000 mg of taurine (Taurine treated group), 300 mg of γ-Oryzanol (γ-Oryzanol treated group) and not treated with any drug (Fat-enriched diet group).
[Diet]
Each group was fed fat-enriched diet. The fat-enriched diet refers to a diet which is obtained by adding 5% cholesterol, 2% cholic acid and 20% beef tallow to a basal diet. The basal diet is obtained from the following composition wherein the vitamin mixture and mineral mixture were the same as described by Harper (J. Nutr., 67, 109 (1959)).
Composition of Basal Diet
______________________________________                                    
Casein          18.0%                                                     
Fat             5.0%                                                      
Vitamin mixture 1.0%                                                      
Mineral mixture 4.0%                                                      
Choline chloride                                                          
                0.2%                                                      
Cellulose       9.6%                                                      
Sucrose         62.2%                                                     
Total           100.0%                                                    
______________________________________
[Experimental Method]
Each group of rats was fed the diet and was bred for 45 days. After the feeding, the blood was collected via the caudal vein of animals to measure the serum cholesterol concentration. After the end of breeding, the liver was excised to measure the cholesterol concentration.
In addition, the mesenteric artery was excised, the surrounding fat was stripped and dyed with Sudan III, and number of fat depositions was counted by microscopy to determine the inhibition rate of fat deposition of the mesenteric artery.
[Results]
The serum cholesterol concentrations are shown in Table 1. The serum cholesterol concentration in Example 1 treated group was kept lower than that in the other groups. The inhibition rate of the liver cholesterol is calculated from the liver cholesterol concentration according to the following formula: ##EQU1##
The liver cholesterol concentration and the inhibition rate of liver cholesterol are shown in Table 2. The liver cholesterol concentration in Example 1 treated group was kept low and the inhibition rate of the fat deposition in Example 1 treated group was high, in comparison with the other groups.
In addition, the deposition of fat was judged by four stages of the number of depositions as 0 to 20, 20 to 100, 100 to 200 and more than 200, which were scored to 1, 2, 3 and 4, respectively, an average score of each group was calculated, and the inhibition rate of the fat deposition of the mesenteric artery in each group is calcurated according to the following formula. ##EQU2##
Table 3 shows average scores of the fat deposition in each group and the inhibition rate of the fat deposition in the mesenteric artery. The inhibition rate of the fat deposition in Example 1 treated group was high in comparison with the other groups.
              TABLE 1                                                     
______________________________________                                    
             Immediately  After   After                                   
Experimental Group                                                        
             after        25 days 45 days                                 
______________________________________                                    
Example 1 treated                                                         
             54.6         115.8   188.1                                   
group                                                                     
Taurine treated                                                           
             55.2         225.5   365.6                                   
group                                                                     
γ-Oryzanol treated                                                  
             53.7         399.4   553.2                                   
group                                                                     
Fat-enriched diet                                                         
             57.8         427.7   767.5                                   
group                                                                     
______________________________________                                    

              TABLE 2                                                     
______________________________________                                    
                Cholesterol                                               
                concentration                                             
                           Inhibition                                     
Experimental Group                                                        
                (mg/g tissue)                                             
                           rate (%)                                       
______________________________________                                    
Example 1 treated                                                         
                26.67      55.9                                           
group                                                                     
Taurine treated 53.33      11.9                                           
group                                                                     
γ-Oryzanol treated                                                  
                44.92      25.8                                           
group                                                                     
Fat-enriched diet                                                         
                60.51      --                                             
group                                                                     
______________________________________                                    

              TABLE 3                                                     
______________________________________                                    
                         Inhibition                                       
Experimental Group                                                        
                  Score  rate (%)                                         
______________________________________                                    
Example 1 treated 0.78   70.8                                             
group                                                                     
Taurine treated   1.20   55.1                                             
group                                                                     
γ-Oryzanol treated                                                  
                  2.21   17.2                                             
group                                                                     
Fat-enriched diet 2.67   --                                               
group                                                                     
______________________________________
Experiment 2: Effect of secretion of fat on Hep G2 cell
[Experimental Group]
T+γ Group is treated with 2.0 mM taurine and 0.05 mM γ-Oryzanol, T group is treated with 2.0 mM taurine only, and C group is untreated with any drug as a control group.
[Experimental Method]
Experiment 2 is carried out by the method described in Current Therapeutic Research, vol. 58, No. 8, 787-795 (1995).
1. Preparation of Experimental Medium and Addition Method of Drug
Dulbecco's Modified Eagle's medium (DMEM) (glucose concentration, 4500 mg/ml) containing 10% lipoprotein deficient serum (LPDS) was used as a basal medium. Taurine was dissolved in phosphate-buffered saline (PBS), sterilized by filtration and added to the basal medium to be made up to the concentration of 2.0 mM to give a test medium. γ-Oryzanol was dissolved in dimethyl sulfoxide (DMSO) to be made up to the concentration of 0.05 mM, and added to the basal medium. Cells cultured in the medium containing PBS (concentration of 1%) were used as a control.
2. Culture of Cells and Recovery of Cells and Medium
Hep G2 cells (Wister Institute) were precultured in DMEM (low glucose, 1500 mg/L) containing 10% FCS, penicillin (1×105 units/L) and streptomycin (100 mg/L), and were cultured at 37° C. in an atmosphere containing 95% air and 5% carbon dioxide. The medium was renewed at an interval of 2 to 3 days. After being confluent, the Hep G2 cells were subcultured as follows: after removing the medium in the dish, the Hep G2 cells were washed quickly with 0.25% trypsin solution (containing 0.1% EDTA), and after addition of a suitable amount of a trypsin solution, incubated at 37° C. for about 5 minutes. Then, a suitable amount of the medium containing 10% FCS was added for termination of the reaction of trypsin, and the Hep G2 cells were suspended well and centrifuged (1000 rpm, 5 minutes) for recovery. The cells were divided into 1/2-1/3 and were cultured by using a DMEM medium containing 10% FCS. The number of the cells was counted using a Sitemeter. The Hep G2 cells were planted in dishes (3.5 cm) (85×104 cells/dish) and were cultured by using a DMEM medium containing 1 ml of high glucose (4500 mg/ml) per dish. The Hep G2 cells were made confluent (about 100×104 /dish), after which the medium was renewed by the experimental medium, followed by culturing for 24 hours. The Hep G2 cells were washed once with PBS, and after addition of 1 ml of PBS, recovered using a policeman. 0.5 μ Ci of [3 H]glycerol or 0.5 μ Ci of [14 C]acetic acid per dish as a radioactive lipid precursor was added before beginning the experiment.
Extraction of Lipid in Hep G2 Cells and the Medium
Lipid in the cells and the medium were extracted according to the method of Bligh and Dyer. The recovered Hep G2 cells were melted and crushed by using a sonicator (Sonifier 250, Bronson Corp.) to give cell homogenate. A part of the cell homogenate was taken into a spitz tube with a screw cap, 3 ml of methanol and 1.5 ml of chloroform were added thereto, followed by stirring well. The mixture was heated at 37° C. for 40 minutes, and 1.5 ml of chloroform and 1.6 ml of water were added thereto to give a mixture wherein the ratio of chloroform : methanol : water was 1:1:0.8, which was then centrifuged (3000 rpm, 15 minutes). The lower-layer chloroform was recovered, concentrated under a nitrogen gas atmosphere and dissolved in a definite amount of petroleum ether, and lipid in the cells was extracted. Lipid in the medium was also extracted in the same manner.
4. Analysis of Lipid
The extracted lipid was fractionated into free cholesterol, cholesterol ester, diglyceride, triglyceride, etc. by using a silica gel G (0.25 mm in thickness)-thin layer chromatography (TLC) developing with petroleum ether : diethyl ether : acetic acid (82:18:1). After completion of the development, each of lipid fractions was detected by coloring with iodide vapor. After the fractionation with TLC, the silica gel of each fraction on the plate was taken into a vial for measurement, dissolved in Sintisol EX-H (Dojin Chemical Co.), and the radioactivity in each fraction was measured with a liquid scintillation counter (Wallac 1410, Pharmacia Co.). Correction was made for the background radioactivity and for quenching by the silica gel.
5. Determination of Cellular Protein
A part of the cell homogenate was used for determination of the protein according to the method of Lowry.
6. Statistical Analysis
Data obtained by the experiment were treated statistically by using Duncan's multiple test.
[Results]
Table 4 shows effect on the excretion of the labeled lipid (cholesterol ester, free cholesterol and triglyceride) after 24 hours by incorporating [14 C]acetic acid into Hep G2 cell lipid medium. The excretion of the labeled lipid in T+γ group was more significantly inhibited than that in the other groups.
Table 5 shows effect on the excretion of the labeled lipid (total lipid, triglyceride and diglyceride) after 24 hours by incorporating [3 H]glycerol into Hep G2 cell lipid medium. The excretion of the labeled lipid in T+γ group was more significantly inhibited than that in the other groups.
              TABLE 4                                                     
______________________________________                                    
               [.sup.14 C] dpm 10.sup.3 /mg                               
Labeled lipid  protein                                                    
______________________________________                                    
Cholesterol ester                                                         
C Group        2.33                                                       
T Group        1.36                                                       
T + γ Group                                                         
               0.93                                                       
Free cholesterol                                                          
C Group        9.65                                                       
T Group        6.43                                                       
T + γ Group                                                         
               4.60                                                       
Triglyceride                                                              
C Group        5.86                                                       
T Group        4.26                                                       
T + γ Group                                                         
               3.78                                                       
______________________________________                                    

              TABLE 5                                                     
______________________________________                                    
              [.sup.3 H] dpm 10.sup.3 /mg                                 
Labeled lipid protein                                                     
______________________________________                                    
Total lipid                                                               
C Group       7.90                                                        
T Group       6.34                                                        
T + γ Group                                                         
              4.30                                                        
Triglyceride                                                              
C Group       2.34                                                        
T Group       1.84                                                        
T + γ Group                                                         
              1.22                                                        
Diglyceride                                                               
C Group       0.17                                                        
T Group       0.05                                                        
T + γ Group                                                         
              0.05                                                        
______________________________________

Claims (4)

What is claimed is:
1. A pharmaceutical preparation for treating or preventing hyperlipemia comprising taurine and γ-oryzanol as effective components.
2. The pharmaceutical preparation of claim 1 wherein the ratio of taurine to γ-oryzanol is within the range of 300:1 to 10:1.
3. A method for treating or preventing hyperlipemia in a mammal which comprises administering to said mammal a pharmaceutical preparation containing taurine and γ-oryzanol as effective components.
4. The method of claim 3 wherein the ratio of taurine to γ-orzyanol is within the range of 300:1 to 10:1.
US09/180,745 1996-05-29 1996-05-29 Remedy or preventive for hyperlipemia Expired - Fee Related US6057308A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP1996/001443 WO1997045112A1 (en) 1995-11-29 1996-05-29 Remedy or preventive for hyperlipemia

Publications (1)

Publication Number Publication Date
US6057308A true US6057308A (en) 2000-05-02

Family

ID=14153344

Family Applications (1)

Application Number Title Priority Date Filing Date
US09/180,745 Expired - Fee Related US6057308A (en) 1996-05-29 1996-05-29 Remedy or preventive for hyperlipemia

Country Status (4)

Country Link
US (1) US6057308A (en)
EP (1) EP0933080A4 (en)
AU (1) AU5844196A (en)
WO (1) WO1997045112A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050182036A1 (en) * 2002-08-02 2005-08-18 Sankyo Company, Limited Medicinal composition containing an HMG-CoA reductase inhibitor

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7037934B2 (en) 2000-12-14 2006-05-02 Sankyo Company, Limited Blood lipid ameliorant composition
WO2002047682A1 (en) 2000-12-14 2002-06-20 Sankyo Company, Limited Blood lipid ameliorant composition
AU2002221130A1 (en) * 2000-12-14 2002-06-24 Sankyo Company Limited Blood lipid ameliorant composition

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CA 118:622, Ito et al., 1992. *
CA 120:297323, Venkatesan et al., 1993. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050182036A1 (en) * 2002-08-02 2005-08-18 Sankyo Company, Limited Medicinal composition containing an HMG-CoA reductase inhibitor

Also Published As

Publication number Publication date
EP0933080A1 (en) 1999-08-04
WO1997045112A1 (en) 1997-12-04
EP0933080A4 (en) 2002-02-27
AU5844196A (en) 1998-01-05

Similar Documents

Publication Publication Date Title
US4602005A (en) Tigogenin cellobioside for treating hypercholesterolemia and atherosclerosis
Kovanen et al. Regulation of low density lipoprotein receptors by adrenocorticotropin in the adrenal gland of mice and rats in vivo.
Melchior et al. Chylomicron metabolism during dietary-induced hypercholesterolemia in dogs.
Quarfordt et al. Metabolism of doubly-labeled chylomicron cholesteryl esters in the rat
CN1452485A (en) Cannabinoid drugs
JPS6038324A (en) Fatty acid composition
EP0488513B1 (en) Use of dioxabicyclo [3.3.0]octane derivatives for the manufacture of a medicament for inhibiting the metabolism of cholesterol.
IL93896A (en) Pharmaceutical compositions containing vitamin d3 derivatives for suppression of parathyroid hormone synthesis and secretion
EP0588621A2 (en) Neutral lipids from endosperm of job's tears and compositions including them
Lovati et al. Increased plasma and aortic triglycerides in rabbits after acute administration of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin
Hall et al. Hypolipidemic activity of tetrakis‐μ‐(trimethylamine‐boranecarboxylato)‐bis (trimethylamine‐carboxyborane)‐dicopper (II) in rodents and its effect on lipid metabolism
US6057308A (en) Remedy or preventive for hyperlipemia
US4812443A (en) Methods for enhancing differentiation and proliferation of hematopoietic progenitor cells
JPH0319206B2 (en)
EP0278681B1 (en) An antihepatopathic composition
DE68911509T2 (en) 26-AMINOCHOLESTEROL AND DERIVATIVES AND ANALOGS THEREOF IN REGULATING CHOLESTEROL accumulation in the body vessels.
Harris et al. Metabolism of griseofulvin in dogs
Packard et al. Effects of 1, 2-cyclohexanedione modification on the metabolism of very low density lipoprotein apolipoprotein B: potential role of receptors in intermediate density lipoprotein catabolism.
WAKASUGI et al. Action of malotilate on reduced serum cholesterol level in rats with carbon tetrachloride-induced liver damage
Hall et al. Hypolipidemic effects of 2‐Furoic acid in sprague‐dawley rats
EP0813866A2 (en) Therapeutic agent for joint diseases
Shohat et al. T lymphocytes and plasma inhibitory factor in ACTH-dependent Cushing's patients
Hall et al. Antihyperlipidemic activity of saccharin analogues in rodents
WAGO Antiatherosclerotic and anticoagulant activity of cerebroside sulfate
Hall et al. The effects of phthalimide and saccharin derivatives on low-density lipoprotein (LDL) and high-density lipoprotein (HDL) receptor activity and related enzyme activities

Legal Events

Date Code Title Description
AS Assignment

Owner name: TAISHO PHARMACEUTICAL CO., LTD., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KITAJIMA, HIDEAKI;TSUNODA, KENJI;YANAGITA, TERUYOSHI;AND OTHERS;REEL/FRAME:010049/0066

Effective date: 19981109

FEPP Fee payment procedure

Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

REMI Maintenance fee reminder mailed
REMI Maintenance fee reminder mailed
LAPS Lapse for failure to pay maintenance fees
FP Lapsed due to failure to pay maintenance fee

Effective date: 20040502

STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362